Acute phase inflammation is characterized by rapid changes in plasma/peritoneal fluid N-glycosylation in mice.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.Murine zymosan-induced peritonitis is a widely used model for studying the molecular and cellular events responsible for the initiation, persistence and/or resolution of inflammation. Among these events, it is becoming increasingly evident that changes in glycosylation of proteins, especially in the plasma and at the site of inflammation, play an important role in the inflammatory response. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based glycosylation profiling, we investigated the qualitative and quantitative effect of zymosan-induced peritonitis on N-glycosylation in mouse plasma and peritoneal fluid. Our results show that both N-glycomes exhibit highly similar glycosylation patterns, consisting mainly of diantennary and triantennary complex type N-glycans with high levels (>95 %) of galactosylation and sialylation (mostly NeuGc) and a medium degree of core fucosylation (30 %). Moreover, MS/MS structural analysis, assisted by linkage-specific derivatization of sialic acids, revealed the presence of O-acetylated sialic acids as well as disialylated antennae ("branching sialylation") characterized by the presence of α2-6-linked NeuGc on the GlcNAc of the NeuGcα2-3-Galβ1-3-GlcNAc terminal motif. A significant decrease of (core) fucosylation together with an increase of both α2-3-linked NeuGc and "branching sialylation" were observed in N-glycomes of mice challenged with zymosan, but not in control mice injected with PBS. Importantly, substantial changes in glycosylation were already observed 12 h after induction of peritonitis, thereby demonstrating an unexpected velocity of the biological mechanisms involved.Dutch Arthritis Association (Reumafonds) LLP-24 Innovative Medicines Initiative Joint Undertaking (IMI JU)/ 115142-2 Netherlands Genomic Initiative/93511033 info:eu-repo/grantAgreement/EC/FP7/278535info:eu-repo/grantAgreement/EC/FP7/27853

Differential mobility separation of leukotrienes and protectins.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageDifferential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to μLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.Prof. Jan Veltkamp fond

Anti-Inflammatory and Proresolving Effects of the Omega-6 Polyunsaturated Fatty Acid Adrenic Acid.

To access publisher's full text version of this article click on the hyperlink belowPolyunsaturated fatty acids (PUFAs) and their metabolites are potent regulators of inflammation. Generally, omega (n)-3 PUFAs are considered proresolving whereas n-6 PUFAs are classified as proinflammatory. In this study, we characterized the inflammatory response in murine peritonitis and unexpectedly found the accumulation of adrenic acid (AdA), a poorly studied n-6 PUFA. Functional studies revealed that AdA potently inhibited the formation of the chemoattractant leukotriene B4 (LTB4), specifically in human neutrophils, and this correlated with a reduction of its precursor arachidonic acid (AA) in free form. AdA exposure in human monocyte-derived macrophages enhanced efferocytosis of apoptotic human neutrophils. In vivo, AdA treatment significantly alleviated arthritis in an LTB4-dependent murine arthritis model. Our findings are, to our knowledge, the first to indicate that the n-6 fatty acid AdA effectively blocks production of LTB4 by neutrophils and could play a role in resolution of inflammation in vivo

Dectin-1/2–induced autocrine PGE₂ signaling licenses dendritic cells to prime Th2 responses

<div><p>The molecular mechanisms through which dendritic cells (DCs) prime T helper 2 (Th2) responses, including those elicited by parasitic helminths, remain incompletely understood. Here, we report that soluble egg antigen (SEA) from <i>Schistosoma mansoni</i>, which is well known to drive potent Th2 responses, triggers DCs to produce prostaglandin E2 (PGE₂), which subsequently—in an autocrine manner—induces OX40 ligand (OX40L) expression to license these DCs to drive Th2 responses. Mechanistically, SEA was found to promote PGE₂ synthesis through Dectin-1 and Dectin-2, and via a downstream signaling cascade involving spleen tyrosine kinase (Syk), extracellular signal-regulated kinase (ERK), cytosolic phospholipase A₂ (cPLA₂), and cyclooxygenase 1 and 2 (COX-1 and COX-2). In addition, this pathway was activated independently of the actions of omega-1 (ω-1), a previously described Th2-priming glycoprotein present in SEA. These findings were supported by in vivo murine data showing that ω-1–independent Th2 priming by SEA was mediated by Dectin-2 and Syk signaling in DCs. Finally, we found that Dectin-2^−/−, and to a lesser extent Dectin-1^−/− mice, displayed impaired Th2 responses and reduced egg-driven granuloma formation following <i>S</i>. <i>mansoni</i> infection, highlighting the physiological importance of this pathway in Th2 polarization during a helminth infection. In summary, we identified a novel pathway in DCs involving Dectin-1/2-Syk-PGE₂-OX40L through which Th2 immune responses are induced.</p></div

SEA promotes PGE₂ synthesis and drives Th2 polarization via signaling through Dectin-1 and Dectin-2 in human moDCs.

<p>(A) cPLA₂ activity 8 h after stimulation. Zymosan was taken along as a positive control for cPLA₂ activation. (B) Protein expression of COX-1 and COX-2 were assessed by western blot. β-actin was used as housekeeping protein. One of 3 experiments is shown. (C) Following 1 h pre-incubation with specific inhibitors for cPLA₂ (Pyr.) or COX-1 and COX-2 (SC and ind., respectively), moDCs were stimulated for 12 h with LPS plus SEAΔα-1/ω-1, and supernatants were collected for PGE₂ determination by LC-MS/MS. (D) At the indicated time points after stimulation with depicted stimuli, phosphorylation of ERK was determined by flow cytometry. A representative flow cytometry plot of intracellular staining for phospho-ERK is shown on the left. (E) PGE₂ levels were determined as in panel C. U0216 was used as inhibitor of ERK. (F) moDCs were treated 45 min with indicated blocking antibodies or isotype controls after which the cells were incubated with PF-647–labeled SEA. Antigen binding/uptake was analyzed by flow cytometry and plotted as relative differences. A representative flow cytometry plot of SEA uptake is depicted on the left. (G) PGE₂ levels were assessed as in panel C, following pre-incubation with blocking antibodies as described in panel F. (H) Syk and (I, J) ERK phosphorylation were determined as described in panel D following pre-incubation with blocking antibodies as described in panel F or panel J with Syk inhibitor R406. Representative flow cytometry plots of Syk (panel H) and ERK (panel I, J) phosphorylation is shown on the left. (K) PGE₂ levels were assessed as in panel C. (L, M) moDCs were pre-incubated with indicated blocking antibodies, followed by 48 h stimulation with LPS plus SEAΔα-1/ω-1, after which OX40L expression was determined by flow cytometry. Data are based on geometric mean florescence. (M) Cells described in panel L were used for T-cell polarization assay as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g002" target="_blank">Fig 2A</a>. Data represent mean ± SEM of 2 (panel D, H, I) or at least 3 independent experiments (panel A, C–G, J–M) and are shown relative to control conditions, which are set to 1 (panel A, C–E, G–M) or 100% (F). “*” and “#”: <i>P</i> < 0.05; “**” and “##”: <i>P</i> < 0.01; “***” and “###”: <i>P</i> < 0.001 for significant differences with the control (*) or between-test condition (#) based on paired analysis (paired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. COX, cyclooxygenase; cPLA₂, cytosolic phospholipase A₂; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; ERK, extracellular signal-regulated kinase; ind., Indometacin; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; moDC, monocyte-derived DC; MR, mannose receptor; OX40L, OX40 ligand; PGE₂, prostaglandin E₂; Pyr., Pyrrophenone; SC, SC236; SEA, soluble egg antigen; Syk, spleen tyrosine kinase; Th2, T helper 2.</p

SEA stimulates PGE₂ secretion and primes Th2 responses independently of ω-1 in human moDCs.

<p>(A) PGE₂ concentration in supernatants from moDC cultures after stimulation with indicated reagents. Concentrations are determined based on an internal standard. Data represent mean ± SEM of 4 independent experiments. (B) moDCs stimulated with indicated lipids (concentration of 2.5 ng/mL for LXA₄, PGE₂, and PGD₂; 12.5 μg/mL for 5-HETE, 8-HETE, and 11-HETE; 25 μg/mL 9-HODE and 13-HODE) were analyzed for Th2 polarizing potential as described in Materials and methods. The ratio of percentage of IL-4⁺ over percentage of IFN-γ⁺ T cells based on intracellular cytokine staining was calculated relative to the control condition. (C) moDCs were pulsed with indicated stimuli and subsequently cocultured with a CD40L-expressing cell line. Supernatants were collected after 24 h, and IL-12p70 concentration was determined by ELISA. (D) T-cell polarization was determined as in panel B. Top and bottom panels show representative flow cytometry plots of intracellular staining of CD4⁺ T cells for indicated cytokines, and the ratio of IL-4 over IFN-γ ratio of these plots based on 4 experiments. Numbers in plots represent frequencies of cells in indicated quadrants. (E) PGE₂ levels as determined by LC-MS/MS in supernatants of moDCs stimulated with indicated stimuli. Data represent mean ± SEM of 3 independent experiments. (A) Statistical significance of different time points per condition compared to baseline (0 h) time point. “*” and “#”: <i>P</i> < 0.05; “**”: <i>P</i> < 0.01. (A) based on two-way ANOVA test or (B–D) for significantly different with the LPS control (*) or between-test conditions (#) based on paired analysis (paired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. CD4, cluster of differentiation 4; HETE, Hydroxyeicosatetraenoic acid; HODE, Hydroxyoctadecadienoic acid; IFN-γ, interferon γ; IL-4, interleukin 4; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; LXA₄, lipoxin A₄; moDC, monocyte-derived DC; PGE₂, prostaglandin E2; SEA, soluble egg antigen; Th2, T helper 2.</p

Th2 polarization induced by SEA is mediated via Dectin-2 and Syk signaling in vivo.

<p>(A–D) WT or CD11c^ΔSyk mice were injected with SEA (panel A, B), SEAΔα-1/ω-1 (panel C), or ω-1 (panel D) in the hind footpad, and draining pLNs were analyzed 7 d later. (A, C, D) pLN cells were restimulated with PMA/Ionomycin in the presence of brefeldin A, and CD4⁺ T cells were stained for indicated intracellular cytokines and percentage cytokine-positive CD4⁺ T cells enumerated. Based on these data, the ratio between the percentage IL-4⁺ over IFN-γ⁺ CD4⁺ T cells was determined as a measure for overall skewing towards Th2. (B) pLN cells were restimulated with SEA for 72 h, and cytokine levels in culture supernatants were determined. (A–D) Bar graphs represent mean ± SEM of 5 to 6 mice per group and are representative of 2 (panel A–C) or 1 (panel D) experiment. (E) BMDCs cultured from BM from WT, Dectin-1^−/−, or Dectin-2^−/− mice were pulsed overnight with SEAΔα-1/ω-1 and injected into hind footpads after which CD4⁺ T-cell responses were analyzed as in panel A. Representative flow cytometry plots of intracellular staining of CD4⁺ T cells are depicted, of which the data are enumerated in bar graphs representing mean ± SEM of 2 independent experiments with 3 to 6 mice per group. (F) SEA binding and uptake was determined as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g004" target="_blank">Fig 4F</a>. (G) ROS production by indicated BMDCs was determined as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g005" target="_blank">Fig 5E</a>, 1 h after stimulation with SEAΔα-1/ω-1. Based on MFI, bar graphs represent fold change relative to control condition, which is set to 1. (H) BMDCs were stimulated as indicated for 18 h after which expression of OX40L was analyzed by flow cytometry. Representative plots are depicted, of which the data are enumerated in bar graphs and shown as fold change relative to control condition, which is set to 1, based on percentage positive cells. (F–H) Bar graphs represent mean of duplicates or triplicates ± SEM of 2 independent experiments. “*”: <i>P</i> < 0.05; “**” and “##”: <i>P</i> < 0.01; “***” and “###”: <i>P</i> < 0.001 for significant differences with the control conditions (*) or between-test condition (#) based on unpaired analysis (unpaired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. ω-1, omega-1; BMDC, bone marrow–derived DC; CD4, cluster of differentiation 4; H2-DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; IFN-γ, interferon γ; IL-4, interleukin 4; MFI, mean fluorescence intensity; nd/pLN, nondraining/popliteal lymph node; OX40L, OX40 ligand; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; SEA, soluble egg antigen; Syk, spleen tyrosine kinase; Th2, T helper 2; WT, wild-type.</p

OX40L is induced by SEA via PGE₂ signaling and is required for Th2 induction.

<p>(A, B) moDCs were stimulated as indicated for 48 h in the presence or absence of neutralizing anti-PGE₂ antibody, after which expression of OX40L was analyzed by flow cytometry. The fold change based on geometric mean fluorescence is shown relative to LPS, which is set to 1 (dashed line). (A) PGE₂ was taken along as positive control for OX40L expression and a representative histogram plot of OX40L expression is shown on the left. (C) T-cell polarization assay as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g002" target="_blank">Fig 2C</a>. Neutralizing OX40L antibody was added during the DC–T cell coculture. Bar graphs represent means ± SEM of at least 6 independent experiments. “*”: <i>P</i> < 0.05: “**” and “##”: <i>P</i> < 0.01 for significant differences with the control conditions (*) or between-test condition (#) based on paired analysis (paired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. DC, dendritic cell; LPS, lipopolysaccharide; moDC, monocyte-derived DC; OX40L, OX40 ligand; PGE₂, prostaglandin E2; SEA, soluble egg antigen; Th2, T helper 2.</p